Generally, this invention relates to a method of determining the concentration of an analyte in a mixture using a spectrographic method of analysis and unique methods of spectral analysis. Specifically, the invention covers the use of Raman spectroscopy, desiccated metal particles, molecular specific coatings, sample containers, and multivariate analysis to determine the concentration of an analyte.
One of the first examples of the importance of chemical analysis in the chemical industry comes from Pliny the Elder (AD 23-79). Pliny the Elder was concerned about contamination of copper sulfate with iron sulfate. Copper sulfate was the principle ore for making copper and bronze. The purity of the copper ore ultimately determined the purity of the copper or bronze. Pliny the Elder found that the extract of gallnuts turned black in the presence of iron sulfate. This simple visual test was the first example of what today is known as Analytical Chemistry.
Since this early work in metallurgical process control the field of analytical chemistry has expanded greatly. The methods of analysis encompass 4 categories. The oldest, represented by Pliny the Elder""s work, is wet chemistry. This method involves mixing chemicals together to observe a quantitative change. The other three categories are more modern and represent an improvement in sensitivity, measurement time, and selectivity over wet chemical methods. These methods are: spectroscopy, chromatography, and electrochemistry.
This invention relates to a spectroscopic analysis known as Raman spectroscopy. Raman scattering involves the inelastic scattering of light by vibrational modes within a molecule. This can be very advantageous as no two molecules exhibit exactly the same Raman spectrum. This makes it possible to distinguish between similar components in a mixture.
This advantage in specificity associated with Raman spectroscopy is overshadowed by an inherent lack of sensitivity. Typically about one in a million photons of light incident on a sample will take the form of Raman scattering. In practical terms Raman is limited to about one part in a thousand detection levels when the analyte is in a matrix.
The Discovery of Surface Enhanced Raman Scattering
Surface enhanced Raman scattering (SERS) like many scientific discoveries, evolved out of serendipitous events. In the early 1970""s electrochemists began using optical methods to study electrode surfaces. Flieschmann and Hendra decided to experiment with Raman spectroscopy as a method of analyzing electrode surfaces. Due to the low sensitivity of Raman spectroscopy they chose silver as the electrode material since it is easily roughened by oxidation-reduction cycles in the presence of chloride. The growth of silver chloride crystals and reduction back to silver leads to a roughened surface with many times the surface area of a smooth polished electrode. This will increase the Raman signal as there are more molecules in the laser beam. They chose pyridine as the probe molecule as it should adsorb through the pyridine nitrogen and it is an inherently strong Raman scatterer. Their experiment was a success. They did not know it but this was the first experiment using SERS. It was not until four years later that this experiment was correctly interpreted. In 1977 Van Duyne at Northwestern University was also trying to study electrodes with Raman spectroscopy. His approach was to use resonantly enhanced molecular probes to overcome the sensitivity problem. He had performed calculations to determine the amount of resonance enhancement needed to observe a monolayer on an electrode. This number was at least 1000 for a strong scatterer like pyridine. This made Flieschmann and Hendra""s results look anomalous. To test if the enhancement was due to increased surface roughness Van Duyne""s student David Jeanmaire tried a milder oxidation-reduction cycle and achieved even stronger signals. This lead to the first announcement of an anomalous phenomena at silver surfaces.
It is now known that the SERS effect arises through an electromagnetic resonance that can occur strongly in noble metal particles and to a lesser extent in some other metals. The resonance occurs because the electrons in the particle are affected by the excitation light to produce a polarization in the particle that makes it more likely to become more polarized. This phenomenon will produce very large electric fields near the particle surface and thus amplify optical events near the surface that are dependent on the electromagnetic field. Raman scattering is just one class of such events. Others might include fluorescence and absorbance. Further, each may be enhanced through a surface phenomenon as in the case of surface enhanced Raman spectroscopy.
While SERS was discovered on electrode surfaces, it is not limited to these. Today SERS is being performed on evaporated metal surfaces, etched metal foils, microlithographically produced surfaces, carefully assembled particle arrays, colloidal suspensions, and with other methods that are capable of producing small submicron sized particles. An excellent discussion regarding aspects and uses of Raman Spectroscopy in a detection context is contained in the document xe2x80x9cMethod and Apparatus for Detection of a Controlled Substancexe2x80x9d, International Application Published Under The Patent Cooperation Treaty (PCT), WO 98/59234, United States National Stage Application No. 09/446,168.
Several problems have plagued the development of SERS into a practical analytical tool. One such problem is the delicate nature of the SERS substrate. The SERS phenomenon is associated with particles or roughness features that are about {fraction (1/10 )} the size of the wavelength of the light used for excitation. Typically this means 40 to 100 nanometers (a nanometer is one billion of a meter). Particles this size are very susceptible to chemical damage, aggregation, and photo damage.
A survey of the different SERS substrates produces one type that stands out with respect to practical analytical chemistry. These are colloidal suspensions. Two significant advantages are found with colloidal suspensions. First, a large volume of colloidal particles can be made at one time. Within this batch of colloids every sample will be identical. This overcomes the irreproducibility of non free floating particulate surfaces. The second advantage is that the colloidal particles are suspended in a solution and therefore tend to be much less susceptible to thermal damage. They also are subject to Brownian motion which tends to continually refresh the particles in the excitation beam, thus eliminating problems with photodegradation of the sample.
In addition to problems with SERS substrate stability and reproducibility an additional factor needs to be included in the analysis. The SERS substrates are typically noble metal particles. The noble metals are aptly named for their ability to resist the aggressions of other materials. In a practical sense this is good for stability of the surfaces, but is impractical in terms of attracting an analyte to the surface. In order for the SERS substrate to act as a tool for detecting an analyte, it must attract the analyte to the surface or in some way be specifically affected by the analyte to show a spectroscopic response.
Initially SERS was seen as advantageous because of its strong enhancement. This invention realizes a different aspect of SERS. The localization of the SERS enhancement near the surface very effectively separates the signal from the analyte that is in close proximity with the surface from analyte or other material in the sample matrix. The locality of the analyte can be used to a strong advantage with respect to the ease of analysis. SERS allows one to measure an analyte in the presence of species that would strongly interfere and cripple other methods of analysis that do not have a localized area of detection.
The problem of inertness with noble metal SERS active surfaces can be overcome with a coating material that attracts or is affected by the analyte. Basically, this combines the strong advantages of an enhanced sensitivity from the SERS substrate with the reactivity or affinity of a molecular specific coating.
Four classes of coatings can be described for a SERS surface. These are passive coatings that can attract the analyte into close proximity of the surface through a chemical affinity for the analyte, the presence of which is detected by its SERS spectrum. Active coatings bind the analyte reversibly and indicate the presence of the analyte through a spectroscopically observable change in their chemical structure. Reactive coatings actually react with the analyte through a covalent bond and create a new species on the surface; this new species is related to the analyte and produces an analyte distinct spectrum for identification and quantitation. The fourth class of coatings are sandwich coatings that bind the analyte and with the addition of a reporter molecule produce a quantifiable signal for the analyte. The latter often consist of immunological coatings with an inherent specificity built into the coating by an organism""s immune response.
A problem that exists when these coatings are combined with a SERS active surface is that the surface becomes less stable. This is particularly true with colloidal suspensions. Colloidal suspensions are stable because the colloidal particles maintain a strong electrical charge through adsorbed ions. In most SERS active systems the colloids are stabilized by the adsorption of citrate ion. This creates a strong net negative charge on the particles and makes them repel each other in solution. Without this net charge the particles would rapidly coalesce into a SERS inactive aggregate of colloidal material.
When coatings are applied to the active colloids it is often impossible to displace the citrate or if the citrate is displaced the colloids begin to aggregate and fall out of suspension in a short time. In some instances it is necessary to add stabilizers of some type to increase the lifetime of the colloids; however, over long periods of time the effectiveness of these stabilizers may be insufficient. It would be commercially desirable to produce a colloidal SERS active system that contains a coating specific for an analyte which had long term stability. Such an invention would have tremendous advantage as a quick and easy to use method for chemical analysis.
The present invention includes a variety of aspects which may be selected in different combinations based upon the particular application or needs to be addressed. In one basic form, the invention discloses the use of Raman spectroscopy to analyze colloidal particles that have been specially prepared to have long term stability and to be sensitive to a specific analyte or group of analytes. A specific advantage of this approach is that the SERS phenomenon exhibits a signal from material localized near the particle surface. This precludes the need for removing excess analyte, impurity, or reagent that indicates the presence of an analyte from the sample mixture. This aspect combined with the aspect of a coated particle with long term stability leads to the invention of a commercially important one-step assay.
This invention includes aspects of colloidal preparations that can be stored for long periods of time and reconstituted to a SERS active suspension. A particularly important aspect of this is the amount of colloid is determined very accurately though a volumetric delivery of known concentration or delivery of a known mass of colloidal suspension. The mass delivery is enabling to an assay since a large mass of diluted colloid can be used to accurately deliver a small amount of colloid into a sample chamber.
The preparation of the colloidal assay potentially includes pretreatment of the sample chamber to prevent the colloidal particles from binding to the surface or each other. This aspect may also include the use of a sample container that naturally possesses the ability to contain the colloids without affecting their ability to be reconstituted.
The configuration of the test materials in the sample container is an important aspect of this invention. Many of the assays covered under this invention will use reagents that should be added in a sequential fashion. This could be carried out with a one-step addition of sample if the different reagents are placed in matrices that control their rate of release, though in many cases controlled release may not be necessary.
The invention also includes aspects of the colloid particle nature that allow a coating to easily displace a prior coating on the colloid formed during preparation that is present for stability.
In keeping with our goal of designing an assay system that has long term stability such that a pretreated assay could be produced for the customer for later use, the invention includes a sample container design that incorporates these features. Assays are typically performed both individually or multiply. Multiple assays have an advantage that many of the steps involved in the assay can be performed in parallel thus decreasing the time of assay. This invention describes a sample chamber that can be easily fabricated in a multisample format. Additionally, as our assay takes special advantage of the SERS effect to produce a one-step assay the sample containers can be sealed to prevent contamination of the sample or, more importantly, prevent potential spread of the sample which may be hazardous to the testing personnel or facility.
Naturally, further objects of the invention are disclosed throughout other areas of the specifications.